Abstracts of Papers Presented at the Eighth Mammalian Genetics And

Genet. Res., Camb.(1998), 72, pp.59–72. Printed in the United Kingdom # 1998 Cambridge University Press 59

Abstracts of papers presented at the eighth Mammalian Genetics and Deelopment Workshop held at the Wellcome Trust Building, Euston Road, London on 19–21 Noember 1997

" # Edited by: ANDREW COPP and DENNIS A. STEPHENSON " Neural Deelopment Unit, The Institute for Child Health, 30 Guilford Street, London WC1N 1EH, UK # The Galton Laboratory, Uniersity College London, 4 Stephenson Way, London NW1 2HE, UK

Sponsored by: The Wellcome Trust, The Genetical Society and B & K Universal Group Limited

cDNA libraries from single human preimplantation Characterization of gene trap integrations expressed in embryos the developing heart

JAMES ADJAYE, ROBERT DANIELS and PETER J. MCLIVE, GURMAN PALL, MARILYN MONK KATHRYN NEWTON and LESLEY M. Molecular Embryology Unit, Institute of Child Health, FORRESTER 30 Guilford Street, London WC1N 1EH, UK Centre for Genome Research, King’s Buildings, West Mains Road, Edinburgh EH93JQ, Scotland We describe the construction, evaluation and application of cDNA libraries from unfertilized oocytes, We describe the characterization of two gene trap and single 2-cell, 4-cell, 7-cell and blastocyst stage integrations (R124 and R213) that were identified as embryos. The corresponding parental samples (cumu- being retinoic acid (RA)-responsive in itro and lus cells and sperm) have also been obtained for the expressed in the developing heart in io [1]. In construction of genomic libraries. The cDNA libraries embryos carrying the R124 integration, reporter gene display sufficient complexities (between 100000 and expression was first detected in the developing heart at 1000000 independent clones) to represent the entire day 8n5 post coitum (p.c.) and was cardiac-specific active gene population at these early stages of human throughout gestation. The majority (70%) of animals development. The ubiquitous cytoskeletal elements, homozygous for this integration die at birth, possibly beta-actin, keratin-18 and alpha-tubulin, were de- due to a right ventricular defect. Surviving homo- tected at the expected frequencies. Sequencing of zygotes subsequently develop defects in both the adult consecutively picked random clones showed the heart and kidney, where the reporter has also been presence of a variety of sequences, such as the human shown to be expressed. Molecular analysis of the transposable element, LINE-1 and Alu repeat sequen- integration site revealed that the vector had integrated ces, the housekeeping gene, HPRT, and tissue-specific into an exon of a novel gene. In embryos carrying the genes alpha-globin and FMR-1. A high proportion of R213 integration reporter gene expression was first novel sequences as well as cDNAs corresponding to detected in the developing heart at day 8n5 p.c. known ESTs (expressed sequence tags) in the Gen- Expression was also detected throughout the yolk sac, Bank and dbEST databases were also detected. detectable in the presumptive yolk sac as early as day Applications of the libraries to several areas of interest, 7n0 p.c. In late gestation and adulthood, expression such as expression of CpG island-containing ‘tissue- broadened to encompass all muscle subtypes, kidney specific’ genes, developmental genes expressed in a and brain. RACE cloning indicates the gene trap stage-specific manner, and a search for monoallelic vector has integrated into a gene homologous to the expression of imprinted genes will be discussed. These human transcription factor gene, TFEB. Homozygous libraries are an invaluable resource for the unlimited mice are viable and fertile; however, detailed mol- analysis of profiles of gene expression at the onset of ecular characterization indicates that the resultant human development, at a time when important and allele is not a complete null. irreversible events occur that affect the genetic potential of the individual.

1. Forrester, L. M. et al.(1996). Proc. Natl. Acad. Sci. USA 93, 1677–1682.

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Identification and characterization of a novel mouse down this critical region, several mouse strains were gene, G90, which maps to chromosome 6 and encodes a established which contain different spr-derived regions putative tumour suppressor of the X chromosome. Using this strategy, DXMit8 could be confirmed to be a critical marker, although " " R. KRAUSE , M. HEMBERGER , WOLFGANG other regions of the X chromosome also seem to be " " " MAYER , U. ZECHNER , H. HIMMELBAUER , involved in the generation of the phenotype. To # " N. BEAUCHEMIN and R. FUNDELE explain the divergence of the phenotype between " Max-Planck-Institut fuW r MolekulaW re Genetik, Berlin, males and females we investigated X-inactivation or # Germay; McGill Uniersity, Montreal, Canada X-reactivation in female interspecific hybrids. Further mapping studies will reveal the other X-chromosomal By subtractive hybridization we have isolated a cDNA loci that are involved in the development of placental clone designated G90 from the small intestine. dysplasia. Expression analysis of G90 revealed high expression in small intestine, testis, kidney and lung. Lower expression was observed in heart, brain, liver, spleen Analysis of differentially expressed genes in mouse and skeletal muscle. A transcript size of 1n5 kb is seen interspecific hybrid placental dysplasia in all tissues; however, in testis a second transcript of " " 1n3 kb was observed. Further analysis provided evi- ULRICH ZECHNER , MYRIAM HEMBERGER , " # dence that in the intestine and testis G90 is expressed WOLFGANG MAYER , ANNIE ORTH , RALF " " only in non-dividing cells. In post-midgestation mouse KRAUSE and REINALD FUNDELE " embryos, G90 is expressed in the cochlea, in the nasal Max-Planck-Institut fuW r MolekulaW re Genetik, Berlin, # epithelium and in restricted areas of the brain. As Germany; Laboratoire Genome et Populations, Uni- assessed by mRNA in situ hybridization, G90 is not ersiteT de Montpellier, France expressed in proliferating cells of the intestinal epithelium and in intestinal adenomas. Sequencing of It has previously been shown that abnormal placental G90 cDNA gave no open reading frame, indicating development (i.e. hyper- and hypoplasia) occurs in that G90 may be a functional mRNA. Mapping of crosses and backcrosses between different mouse G90 showed that it is located on mouse chromosome (Mus) species. The precise genetic basis for these 6, close to the imprinted gene Mest. Subsequent placental malformations has not been determined. analysis of the imprinting status revealed a strain- However, a locus that contributes to the abnormal specific silencing of one allele in interspecific hybrids, development (Ihpd: interspecific hybrid placental but influence of parental sex could not be found. dysplasia) has been mapped to the X chromosome. We applied subtractive hybridization to isolate Genetic analysis of placental dysplasia in mouse cDNAs differentially expressed in normal and hyper- interspecific hybrids plastic placentas. In the first experiment, several cDNAs representing known genes as well as seven " " MYRIAM HEMBERGER , ULRICH ZECHNER , novel mouse cDNAs were identified. Further charac- " " WOLFGANG MAYER , RALF KRAUSE , terization of these cDNAs is in progress. Previous " # MATTHIAS REULE , BERNHARD KORN , studies in interspecific hybrids of the rodent genus $ ROSEMARY ELLIOTT , ANNEMARIE Peromyscus (‘deer mice’) described similar placental # " POUSTKA and REINALD FUNDELE phenotypes that are correlated with an altered genomic " Max-Planck-Institut fuW r MolekulaW re Genetik, Berlin, imprinting of several genes. In our laboratory the # Germany; Deutsches Krebsforschungszentrum, expression of imprinted genes such as Mash2, Igf2 and $ Heidelberg, Germany; Roswell Park Cancer Institute, H19 in placentas of Mus interspecific hybrids was Buffalo, NY, USA determined by RNA in situ and Northern analysis. To verify the imprinting status of these genes in Mus Placental dysplasia was observed in interspecific interspecific hybrids, we are now investigating parental hybrids between Mus musculus (mus) and M. spretus allele-specific expression by RT-PCR. (spr). When (M. musiM. spr)F"females were mated with M. mus males, placental hyperplasia was observed, whereas placental hypoplasia occurred in backcrosses with M. spr males. Both forms of placental dysplasia were more prominent in males than in females. One of the loci contributing to placental dysplasia, Ihpd (interspecific hybrid placental dysplasia), was mapped to the central portion of the mouse X chromosome near DXMit8. To narrow

Characterization of a novel mouse gene with homologues sequence comparison between the mouse and human on the X and Y chromosomes genomes. A minimal tiling path of cosmid and BAC clones covering nearly 600 kb of the Ids–Dmd region " " " P. ELLIS , I. EHRMANN , D. SCOTT ,E. is available for sequencing plus some existing sequence " # # SIMPSON , N. BROCKDORFF , S. DUTHIE ,S. which has already been analysed. At current through- $ $ MAZERAT and M. MITCHELL put, we expect by early 1998 to have completed and " # Transplantation Biology Group, X Inactiation analysed 600 kb of sequence. Fingerprinting and Group, MRC Clinical Sciences Centre, Imperial College generation of cosmid\BAC tiling paths is being carried $ School of Medicine, London, UK; INSERM UniteT out at the MGC and Ohio State, USA. Sequencing is 406, Marseille, France being carried out at the HGMP-RC, while further computing analysis, including exon prediction, is The genes encoding the male-specific minor histo- performed at the MGC. compatibility antigens are located on the short arm of the mouse Y chromosome, in the Sxrb deletion interval. Two such genes, Smcy and Uty, have recently Mutation analysis of mottled mice been identified that encode H-YKk and H-Ydb epitopes respectively. We have cloned another gene from this PAMELA CUNLIFFE, VIVIENNE REED, region, Tfy, which lies between Smcy and Uty and is WALTER MASSON and YVONNE BOYD homologous to a translation factor. We have also MRC Mammalian Genetics Unit, Harwell, Didcot, shown that there is an X chromosome homologue of Oxfordshire OX11 0RD, UK this gene, as well as pseudogene copies on the X and chromosome 15 in some mouse strains. The predicted Mottled mice and Menkes disease patients have X- translation products of the X and Y structural copies linked conditions caused by aberrant handling of differ by only 10 amino acids and both copies are cellular copper. Menkes disease patients classically widely transcribed; the Y copy is therefore a candidate follow a severe course, dying before 3 years of age. for H-Y. The X copy maps close to the Dmd locus and However, there are milder forms of the disease, is expressed from the inactive X chromosome, including cutis laxa. There are over 20 alleles of indicating that the expression of two copies of this mottled and these can be grouped into three classes gene is required in both males and females. based on the phenotype of the affected males. Males either die before birth, die within a few weeks of birth or survive to adulthood. Mutations in a gene encoding a copper-transporting ATPase (ATP7A) cause Comparative sequencing on the mouse and human X Menkes disease. The first mutation found in the chromosomes homologous gene in mouse (Atp7a) was a splice site " # change in mottled blotchy. This is an allele in which M. R. M. BOTCHERBY , M. A. STRIVENS ,R. " " " affected males survive to adulthood and may be a STRAW , G. W. WILLIAMS , S. FERNANDO , # " good model for cutis laxa. Subsequently, we and A. M. MALLON , Y. UMRANIA ,P.M. " " # others have discovered mutations in mottled alleles of WOOLLARD , M. GILBERT , R. BATES ,K. " " # other classes. These range from single base pair GOODALL , J. S. GREYSTRONG , P. DENNY , " " $ substitutions to deletions of several kilobases. Gen- M. RHODES , C. R. MUNDY , G. E. HERMAN # erally, the severity of the genetic lesions correlates well and S. D. BROWN " with that of the phenotype. However, our studies on UK Human Genome Mapping Project Resource the kinetics of copper processing in cell lines from a Centre, Hinxton Hall, Hinxton, Cambridgeshire CB10 # number of mottled alleles suggest that there is no 1SB, UK; MRC Mouse Genome Centre, Harwell, $ signifcant difference between alleles at this level. Didcot, Oxfordshire OX11 0RD, UK; Department of Pediatrics and Children’s Hospital Research Foun- dation, Ohio State Uniersity, Columbus, OH 43205, USA Genetic and developmental studies of neuronal mi- gration defects in the dreher mouse We aim to sequence 3 Mb from the Ids–Dmd region of the mouse X chromosome. This region is homologous CRISTINA COSTA and ANDREW J. COPP to regions Xq28 and Xp21 on the human X Neural Deelopment Unit, Institute of Child Health, 30 chromosome and as such crosses an evolutionary Guilford Street, London WC1N 1EH, UK breakpoint. Xq28 is a gene-rich region receiving considerable attention from sequencing groups and Dreher (dr) is an autosomal recessive mutation causing there will therefore be abundant opportunity for deafness and circling behaviour in homozygotes and,

in some cases, white belly spots. Homozygotes also cycle. Thymidine is available from two sources: the have misplaced neurons (heterotopia) in the CNS that kinase pathway uses deoxyuridine monophosphate involve both the cerebral hemispheres and the cer- (dUMP) and folate coenzymes to synthesize deoxy- ebellum. Genetic mapping of dreher with respect to thymidine monophosphate (dTMP), whereas the DNA microsatellite markers led to the positioning of salvage pathway uses thymidine kinase to reutilize the dreher gene in a 5n78 cM interval on distal mouse thymidine. In the normal condition, dUMP suppresses $ chromosome 1, proximal to its previously assigned the uptake of [ H]thymidine. If folate metabolism is $ location. The study also provided flanking markers perturbed, however, the uptake of [ H]thymidine is for dreher that enabled precise genotyping of pre- and increased, as a greater percentage is used from the post-natal individuals in all subsequent studies. Two salvage pathway. We found that embryos cultured in genes that map to the dreher region were evaluated as the presence of folate antagonists such as 5-fluoro- candidates for the mutation: Astrotactin and Pou2f1. uracil and cycloleucine have a deranged suppression There were no differences in the mRNA expression test, as predicted. The test was then applied to mutant pattern of Astrotactin between dr\dr and j\j mice which develop NTD. Embryos homozygous for littermates between E9n5 and E11n5, making Astro- either looptail (lp) or curly tail (ct) showed normal tactin an unlikely candidate, although not definitely deoxyuridine suppression tests suggesting that there is ruling it out. Sequencing of the POU-specific domain no defect in folate metabolism in either mutant. of Pou2f1 did not show any mutations in dr\dr Homozygous splotch (Sp2H) embryos, which develop animals. Howerver, whole-mount in situ hybridization exencephaly and\or spina bifida, showed an abnormal of embryos showed a spatially more restricted ex- suppression test, suggesting that these embryos have pression of Pou2f1 at the hindbrain–midbrain bound- an abnormality of folate\homocysteine metabolism. ary in dr\dr compared with wild-type littermates at In itro and in io treatment of homozygous splotch E11n5, suggesting that this gene may be primarily or embryos with folic acid or thymidine reduced the secondarily affected in dreher. The neocortex of dr\dr incidence of both cranial and caudal NTD whereas mice exhibts a focal or diffuse increase in the neuronal treatment with methionine was found to increase the density of layer I. Birthdating studies were performed incidence of NTD. These findings suggest that splotch using bromodeoxyuridine labelling in io. The het- embryos have a defect within the folate cycle per se, erotopic neurons appear to be generated throughout rather than in the associated homocysteine–methio- the period of neocortical histogenesis, not pre- nine cycle, and that the splotch mutant provides a dominantly at the start of neurogenesis, as would be model for analysis of folate-preventable NTD. predicted if they were derived from the preplate. Moreover, excessive proliferation of early-born cells (i.e. those destined for layer I) is unlikely to be a Genetic and physical mapping around the mouse epilepsy contributing factor in the production of heterotropia locus, lethargic (lh) in dreher. Histological studies reveal abnormalities of the neocortical glial limiting membrane in dr\dr mice. NICHOLAS PARKINSON, JANE BARCLAY, This defect may play a central role in the genesis of MARK GARDINER and MICHELE REES heterotopic layer I neurons. Department of Paediatrics, Uniersity College London Medical School, Rayne Institute, 5 Uniersity Street, London, UK Detection of disturbed folate metabolism in mouse embryos developing neural tube defects The lethargic trait is an autosomal recessive single locus mouse epilepsy mutant, linked to chromosome ANGELEEN FLEMING and ANDREW J. COPP 2. Recently the locus has been cloned and identified as Neural Deelopment Unit, Institute of Child Health, 30 a mutant splice variant of the voltage-dependent Guilford Street, London WC1N 1EH, UK calcium channel beta 4 subunit (Cacnb4). Initially a fine-scale genetic mapping of this locus was created The role of folate in the prevention of human neural using two crosses as the mapping resource. Marker tube defects (NTD) is well established, although little typing of over 1000 meioses with a range of is known about the mechanism by which peri- polymorphic loci on mouse chromosome 2 narrowed conceptional supplementation produces this effect. In the lethargic region to approximately 2n0 cM. A order to study the action of folate experimentally, we chromosome walk was initiated across the shortest adapted the deoxyuridine suppression test for use in recombinant region using the flanking markers as whole embryo cultures, allowing the efficiency of the starting points for isolation of YAC clones. Sequence folate cycle to be assayed during the period of tagged site and simple sequence length polymorphism neurulation. The test uses thymidine incorporation as capturing techniques have been implemented on a measure of the efficacy of the folate\homocysteine existing YAC clones allowing the extension of the two

contigs. Presently a total estimated physical distance Renal agenesis in mice homozygous for a gene trap of 4 Mb has been mapped. This project initially aimed mutation in the gene encoding heparan sulphate 2- to isolate and characterize the lethargic locus in sulphotransferase mouse. With the elucidation of lh via a candidate gene " # approach, focus has shifted towards assessing this SIMON BULLOCK , JUDY FLETCHER , # gene’s possible role in analogous human disorders. VERONICA VAN HEYNINGEN , ROSA " # Work will continue to reﬁne these physical and genetic BEDDINGTON and VALERIE WILSON " maps, providing a substantial resource for workers Laboratory of Mammalian Deelopment, MRC concerned with the same region. National Institute for Medical Research, The Ridgeway, # Mill Hill, London NW7 1AA, UK; MRC Human Genetics Unit, Western General Hospital, Crewe Road, Diﬀerential display analysis of dissected mouse somites Edinburgh EH4 2XU, Scotland

RAJEEV GUPTA, DENNIS SUMMERBELL and Heparan sulphate proteoglycans (Hspgs) are expressed PETER W. J. RIGBY at the surface of most cells in multicellular organisms Diision of Eukaryotic Molecular Genetics, National and have been implicated in the presentation of a Institute for Medical Research, The Ridgeway, Mill number of secreted signalling molecules, including Hill, London NW7 1AA, UK members of the fibroblast growth factor (FGF), Wingless\Wnt and transforming growth factor-beta Conventional techniques of screening for differential (TGF-β) families to their signal-transducing receptors. gene expression have involved the construction of The specificity of Hspg ligand interactions resides, at libraries and subsequent differential or subtractive least in part, in the structure of the heparan sulphate screening. In the mouse embryo, where tissue avail- side chains, which vary in number, length, sequence ability is limited, such methods are difficult. Differ- composition and sulphation pattern between cell type ential display provides a rapid alternative approach to and developmental stage. Thus the production of this problem. However, conventional display proto- differentially glycosylated proteoglycans may rep- cols give differential clones that are unsuitable for resent an additional means to regulate cell–cell secondary screening by in situ hybridization and have communication during development. In an attempt to high false positive rates. We have been using our own identify molecules important for mouse embryogenesis differential display protocol to isolate novel genes we have employed a gene trap strategy that identifies, related to somitogenesis. We observe that using the mutates and reports on the embryonic expression primers of known genes and poly A-enriched RNA pattern of genes expressed in murine embryonic stem from individual dissected mouse somites and preso- cells. We have characterized a mutation in the gene mitic mesoderm, predicted patterns of gene expression encoding heparan sulphate 2-sulphotransferase. This can be detected by RT-PCR. Subsequently we have gene is expressed differentially during embryogenesis, generated reproducible RT-PCR fingerprints using presumably directing changes in proteoglycan side arbitrary primers from which we have been able to chain structure. Moreover, mice homozygous for the clone novel cDNA fragments large enough for rapid gene trap mutation exhibit bilateral renal agenesis, secondary screening using in situ hybridization. Using resulting from a failure of ureteric bud branching, as this approach, we have isolated a number of novel well as defects of the eye and skeleton. These data genes, including a family of transmembrane genes provide the first genetic demonstration of HSPG containing leucine-rich repeat motifs related to those function in vertebrate embryonic development. of the Drosophila genes Slit and Tartan, which have interesting patterns of expression in the somites.

New Wilms’ tumour suppressor gene (WT1) functions were designed to study gene expression and regulation revealed by YAC transgenic analysis at the onset of embryonic genome activity. Inter- crossing of heterozygotes from the F27 line did not " " " A. MOORE , A. SCHEDL ,L.MINNES ,J. produce any liveborn homozygotes. These observ- # $ " KREIDBERG , R. JAENISCH and N. HASTIE ations are typical of a recessive insertional mutation " MRC Human Genetics Unit, Western General Hos- responsible for the in utero death of homozygous pital, Crewe Road, Edinburgh, EH4 2XU, UK; mutant embryos. Analysis of F27 embryos at various # Diisions of Nephrology and Newborn Medicine, times during gestation shows that homozygous trans- Children’s Hospital, Department of Pediatrics, Haard genic embryos die between the beginning of embryonic Medical School, 300 Longwood Ae, Boston, MA development and the 14th day post coitum. They $ 02115, USA; Whitehead Institute for Biomedical showed extensive external and internal abnormalities Research\Nine Cambridge Center, Cambridge, MA (e.g. haemorrhagic regions, misdeveloped organs). 02142-1479, USA Molecular and genetic aspects of this mutation are now under investigation. FISH analysis established We have used YAC transgenic technology to analyse that the transgene was located in the proximal region the Wilms’ tumour gene (WT1) function and ex- of chromosome 9. Flanking sequence to the transgene pression. Disruption of WT1 predisposes to childhood has been cloned. Analysis of these sequences failed to kidney neoplasia, leukaemia and gonadal dysgenesis. identify a candidate gene. However, we were able to Homozygous Wt1 ‘knockout’ mice die at around 13n5 identify homology with some new human and mouse days of gestation showing no development of kidneys DNA gene sequences. (Acknowledgement:C.P.isthe or gonads and disruption of thoracic organs. We recipient of Fonds pour la formation la Recherche isolated a 470 kb YAC carrying the human WT1 dans l’Industrie et dans l’Agriculture studentship.) locus centrally. This was then truncated 3h of WT1 giving a 280 kb YAC. Expression of the 280 kb YAC Effects of thyroid hormone deficiency on mice selected transgene on the Wt1 ‘knockout’ background rescued for increased and decreased body weight and fatness the thoracic defects which cause embryonic lethality. " # However, neonates died within 48 h lacking kidneys, LUTZ BUNGER , HELEN WALLACE , JOHN O. $ " gonads and adrenal glands. Therefore WT1 is required BISHOP , IAN M. HASTINGS and WILLIAM G. " for adrenal, in addition to urogenital, development. A HILL " subset of these partially rescued mice develop kidneys Institute of Cell, Animals and Population Biology, # which arrest at the S-shaped stage of nephrogenesis; Uniersity of Edinburgh, Scotland; Roslin Institute, $ hence WT1 has a role in the maturation of the Roslin, Midlothian, Scotland; Centre for Genome glomerular podocyte cells. We inserted a lacZ reporter Research, Uniersity of Edinburgh, Scotland gene into WT1 exon1 of both the 470 and 280 kb YACs. Expression of the reporter gene in transgenic A study was undertaken to test whether the mice carrying either of these constructs mirrors the elimination of metabolic pathways, which might, a complex endogenous WT1 expression pattern. Fur- priori, be expected to be strongly involved in the thermore transgene activity indicates potential new selection for growth or fatness, comprising thyroid domains of Wt1 function in epaxial musculature, hormones (TH) and growth hormone (GH), is interdigital limb mesenchyme and the nervous system. responsible for a substantial part of the genetic change produced by selection. Mouse lines used in this study Characterization of a recessive insertional mutation have been selected (for c. 50 generations) for high and implying a late embryonic lethality low body weight and for high and low fat content, producing a 3-fold difference in body weight and a 5- " " C. DEPIERREUX , E. CHRISTIANS ,J. fold difference in fat content. Thyroid ablation was " " # MARCHANDISE , M. MINNE , M.-G. MATTII achieved by repeated backcrossing into the selection " and C. DESSY-DOIZI lines a transgene comprising the HSV1-tk gene coupled " Serice d’Histologie-Embryologie, FaculteT de MeT de- to the promoter of the thyroglobulin gene. In the cine VeT teT rinaire, UniersiteT de LieZ ge (Sart-Tilman),B- absence of TH and GH lines still differ in body # 4000 LieZ ge, Belgium; FaculteT de MeT decine de la weight from 10 days to about 100 days. The effect of Timone, INSERM U 406, F-13385 Marseille cedex 05, the transgene was dependent on the genetic back- France ground for almost all body weights and relative gonadal-fat-pad weights, but less for fat content. The Several transgenic mouse lines were created by data show that TH- and GH-status is not the only microinjection of a HSP70.1Luc transgene placing the cause for line differences in growth and fatness, firefly luciferase reporter gene under the control of a caused by long-term selection, but both are involved murine heat shock promoter (HSP70.1). These lines to a significant but neverthelss relatively small extent.

Therefore these results support a polygenic model of has been shown to encode peptides that express the selection response. mouse H-YKk epitope and two human H-Y epitopes that are expressed in association with the HLA B7 Radiation sensitivity in male mouse germ cells and A2 class I molecules. These peptides have been identified. Smcy was also found to express the H-YDk " " Smcy 1 JULIA BROWN , YURI E. DUBROVA , MARK epitope: cells expressing the cosmid (cMEM 4) # " H YDk PLUMB , PHILIPPE BOIS and ALEC J. stimulated an - -restricted T cell clone. The " JEFFREYS expression of this epitope was further localized using " Department of Genetics, Uniersity of Leicester, subcloned genomic fragments and nested sets of PCR- # Leciester LE1 7RH, UK; Radiation and Genetic cDNA products of Smcy,toa310 bp fragment. Stability Unit, Harwell, Oxfordshire OX11 0RD, UK Comparison of the sequences of Smcy and Smcx, the X chromosome homologue of this gene, within this Experiments used to examine radiation-induced mu- fragment identified six amino acid differences between tation in mice have typically employed the specific the two genes. Four synthetic peptides that spanned locus test and have used tens of thousands of mice due these differences were tested and one was identified H YDk to the low frequency of germline mutation at single that stimulated the - -specific T cell clone at 1  copy genes [1]. Minisatellites are tandem repeat arrays concentrations of µ . Two shorter peptides were 1 that show a very high spontaneous mutation rate, synthesized from this 2mer sequence to identify the H YDk thus providing a very efficient monitoring system for optimal peptide that could express the - epitope. germline mutation. Recent studies have shown that acute doses of ionizing gamma-radiation to male mice The Y* rearrangement in mice: new insights into a cause a significant increase in minisatellite mutation perplexing pseudoautosomal region rate [2, 3]. We have extended this analysis, using male " PAUL S. BURGOYNE , SHANTHA K. CBA\H mice, to ascertain the stage of sperma- " # MAHADEVAIAH , JO PERRY , STEPHEN J. togenesis that is sensitive to acute gamma-radiation, # # PALMER and ALAN ASHWORTH establish dose–response curves, estimate the doubling " Laboratory of Deelopmental Genetics, National dose and compare the effects of chronic versus acute Institute for Medical Research, The Ridgeway, Mill radiation exposure on minisatellite loci. Our current # Hill, London NW7 1AA, UK; Chester Beatty Labora- data suggest that mutation induction is only at- tories, Institute for Cancer Research, Fulham Road, tributable to the spermatogonia stage of sperma- London SW3 6JB, UK togenesis, providing evidence for a meiotic origin of murine minisatellite germline mutation. Frequency of mutations induced by acute gamma-radiation in- The Y* chromosome, in essence, is a Y chromosome hijacked by a non-Y centromere attached to the creases linearly with dosage from 0n5Gyto1Gy with pseudoautosomal region (PAR). All the Y-specific the doubling dose between 0n5 Gy and 0n8 Gy. These values are close to those obtained in mice using other region appears to be unaltered, but the pairing monitoring systems [1]. The application of these data behaviour of Y* with the X during male meiosis led to for analysis of radiation-induced mutation in humans the conclusion that part of the PAR is inverted. [4] will be discussed. Recombination between the X PAR and this inverted PAR region produces two recombinant products; one is an X-attached-Y chromosome and the other is a 1. Luning, K. G. & Searle, A. G. (1971). Mutat. Res. 12, 291–304. small chromosome comprising the recombinant PAR 2. Dubrova, Y. E. et al.(1993). Nature Genet. 5, 92–94. attached to the non-Y centromere. Recently, we 3. Fan, Y. J. et al.(1995). Int. J. Radiat. Biol. 68, introduced the X-linked mutation Paf into XY* males 177–183. and to our surprise found that the XPaf Y* males 4. Dubrov, Y. E. et al.(1996). Nature 380, 683–686. were phenotypically wild-type. We have subsequently established that this is because the Y* chromosome Identification of H-Y epitopes in Smcy carries an X PAR boundary together with the wild- type allele of Paf; this X PAR boundary is transferred " " " D. SCOTT , I. EHRMANN , P. ELLIS ,E. to the small recombinant product. We hypothesized " # SIMPSON and M. MITCHELL that the Y* PAR is flanked by a Y PAR boundary " MRC Clinical Sciences Centre, Imperial College and an X PAR boundary (in the opposite orientation); School of Medicine, London W12 0NN, UK; this predicts that there are two regions of proximal # INSERM UniteT 406, Marseille, France PAR running towards each other and raised the possibility that there is no distal PAR. Steroid sulfatase The Smcy gene maps to the short arm of the mouse Y (Sts) is a marker for the distal PAR; by PCR and chromosome into the Sxrb deletion interval. This gene Southern analysis we have been able to show that Sts

is missing from the Y* chromosome. The recom- genetic analysis of such samples is severely limited bination event producing the X-attached-Y chromo- because there is often insufficient DNA to perform some recreates the same PAR structure as in Y*. more than one PCR amplification. We have investigated four different methods of whole genome amplification which generate enough DNA for several Meiotic pairing, non-disjunction and germ cell death in subsequent PCR amplifications to be performed. wild and laboratory-bred male house mice (Mus Indeed, we have demonstrated that in some instances musculus domesticus) carrying Robertsonian trans- over 100 independent PCR amplifications can be locations performed on DNA from a single cell. Cytogenetic " # analysis is also problematic at the single cell level, B. M. N. WALLACE , J. B. SEARLE and C. A. $ mainly because of difficulties in obtaining metaphase EVERETT " chromosomes. Interphase cytogenetics solves some of School of Biological Sciences, Uniersity of Birming- # these problems, but accuracy is reduced when more ham, Birmingham B15 2TT, UK; Department of than five chromosome pairs are analysed due to Biology, Uniersity of York, PO Box 373, York YO1 $ overlapping signals. However, cytogenetic investi- 5YW, UK; Department of Obstetrics and Gynae- gation using comparative genomic hybridization cology, Uniersity of Edinburgh, 37 Chalmers Street, (CGH) overcomes these limitations and can now be Edinburgh EH3 9EW, Scotland performed on single cells following whole genome amplification. Furthermore, CGH can be performed A series of investigations has been carried out on both in addition to numerous specific PCR amplifications, feral and laboratory-bred male house mice (Mus allowing simultaneous molecular and cytogenetic musculus domesticus) to examine the effect of Robert- analysis of the same cell. sonian rearrangements on meiotic chromosome pairing and on the frequency of non-disjunction and germ Checking the stability of human bone marrow stem cell cell death. In mice from a hybrid zone in the north of genome by RAPD-PCR (random amplified polymophic Scotland prophase pairing was orderly and the level of DNA) univalence and germ cell death low. Laboratory-bred animals heteroxygous for the Robertsonian trans- D. ZAKOVA, P. NURENBERG and R. BRDICKA locations Rb(1.3)1Bnr, Rb(11.13)4Bnr and Rb(10.11)- Institute of Haematology, Prague, Czech Republic, and 8Bnr had an increased frequency of pachytene pairing ChariteT Clinic, Berlin, Germany abnormalities and germ cell death over homozygotes. The homozygotes had a low level of non-disjunction To what extent is the genome of human bone marrow but substantial germ cell death which could not be stem cells resistant to the treatment regimen of attributed to pairing problems as these showed no autotransplanted oncology patients? The answer to increase above the level in controls. It was concluded this question is especially important now, since several that both genic factors and pairing problems are retransplantations have been suggested to improve involved in promoting germ cell death and non- the prognosis of patients. In our study we compared disjunction. Their influence on meiosis is being blood samples taken before (A) and approximately 1 investigated further. month after transplantation (B); occasionally a third sample from the frozen cells used for transplantation Cytogenetic and molecular genetic techniques for the was also examined (C). A few patients (5) were studied analysis of single cells by classical DNA fingerprinting in a pilot study conducted by P. Nurenberg, but due to the limited " " DAGAN WELLS , JON SHERLOCK , ALAN amount of DNA (sample B), the method of RAPD # " HANDYSIDE , MATTEO ADINOLFI and JOY with analysis of PCR products by capillary electro- " DELHANTY phoresis was adopted (30 patients). No differences " The Galton Laboratory, Uniersity College London, 4 between samples A and B studied by P. Nurenberg # Stephenson Way, London NW1 2HE, UK; St Thomas’ were observed; RAPD analyses also gave similar Hospital, Lambeth Palace Road, London SE1 7EH, results. In comparison with classical DNA finger- UK printing based on production of different restriction fragments hybridizing with repetitive sequences An increasing number of genetic analyses are being (GTG,CA) and showing polymorphism, the one- performed on minute samples of tissue or DNA. primer RAPD gave nearly identical fragment pattern Examples include preimplantation genetic diagnosis, in all patients. Quantitative differences observed in analysis of fetal cells isolated from the maternal capillary electrophoretograms most probably resulted circulation, forensics, investigation of ancient DNA from the sensitivity of RAPD to the working and analysis of individual tumour cells. Molecular conditions, which were never absolutely identical.

Steady-state levels of subunits of respiratory chain enzymes, NAT1, is expressed in many tissues, in- enzymes: clues to the primary genetic defect in cluding first trimester placentas. The other isoenzyme, cytochrome c oxidase deficiencies NAT2, is approximately 1000-fold less active in placentas. Using heterologously expressed human " " SION WILLIAMS , J. W. TAANMAN ,H.R. NATs, it has been shown that NAT1, but not NAT2, # " SCHOLTE and A. SCHAPIRA metabolizes the folate catabolite p-aminobenzoyl- " Department of Clinical Neurosciences, RFHSM, glutamate (pabaglu) to the N-acetylated form, N- # Rowland Hill Street, London NW3 2PF, UK; Depart- acetylpabaglu [1, 2]. The acetylated form of pabaglu ment of Biochemistry, Erasmus Uniersity, Rotterdam, (N-acetylpabaglu) is the major urinary folate catabo- The Netherlands lite and its excretion increases during pregnancy. Therefore NAT1 is likely to have a role in endogenous Cytochrome c oxidase (COX) is the terminal enzyme metabolism. Using placental homogenates and paba- complex of the mitochondrial respiratory chain (RC). glu as substrate, we have demonstrated that N- In common with two of the three other respiratory acetylpabaglu is formed. The formation of N-acetyl- chain enzymes COX is composed of subunits encoded pabaglu is inhibited by the NAT inhibitor 5-iodosali- by mitochondrial DNA (mtDNA) and subunits of cylate. The activity of NAT1 in placenta is sufficient nuclear origin. COX deficiencies show a remarkable to account for the excess production of N-acetyl- clinical heterogeneity, with different tissues affected pabaglu during pregnancy in addition to its role in and with variable onset of symptoms. In patients with metabolism of environmental arylamines. NAT1 a childhood or late onset, partial defects of COX are shows functional and structural polymorphism, and often associated with specific mutations in a sub- the inter-individual variation in NAT1 activity in population of mtDNA. In paediatric patients, how- placentas may affect folate metabolism during preg- ever, little is known about the molecular basis of the nancy. disease. Cultured fibroblasts from a group of five infants with COX deficiency have been studied. Southern blot hybridization did not reveal any 1. Minchin, R. F. (1995). Biochem. J. 307, 1–3. mtDNA abnormalities. On Western blots, we found 2. Ward, A., Summers, M. J. & Sim, E. (1995). Biochem. Pharmacol. 49, 1759–1767. reduced steady-state levels of both mtDNA-encoded and nuclear-encoded COX subunits. In addition one patient showed reduced levels of subunits of another RC enzyme of dual genetic origin (cytochrome c MBL gene mutations in a large prospective study of reductase), often indicative of a mutation in a mtDNA childhood disease tRNA gene. In another patient, respiratory enzymes " # of dual genetic origin and the single exclusively RENATA M. J. HAMVAS , , MALCOLM W. " # nuclear-encoded RC enzyme (succinate dehydrogen- TURNER , MARCUS PEMBREY , RICHARD $ " ase) were affected. Here a mutation in a common JONES , DOMINIC JACK and the ALSPAC study % & nuclear factor, possibly involved in mitochondrial team , " # import, is suspected. Immunobiology Unit, Mothercare Unit of Clinical $ Genetics and Foetal Medicine, and Chemical Path- ology Unit, Camelia Botnar Laboratories, Institute of Child Health, Guilford Street, London WC1N 1EH, % & Arylamine N-acetyltransferase in human placenta: a UK; Southmead Hospital, Bristol, UK; Diision of role in folate catabolism Paediatric and Perinatal Epidemiology, Institute of Child Health, Bristol, UK " # " V. A. SMELT , H. MARDON , R. DELGODA , $ " % " L.-Y. LIAN , J. SINCLAIR , R. APLIN and E. SIM The Avon Longitudinal Study of Pregnancy and " Department of Pharmacology, Mansfield Road, Childhood (ALSPAC or Children of the Nineties) was # Oxford OX1 3QT, UK; Biological NMR Centre, set up as a multi-faceted study on 14000 children born Uniersity of Leicester, Leicester LE1 9HN, UK; in the Avon area from 1991 to 1992. The aim of the $ Department of Obstetrics and Gynaecology, John project is to examine the origins and influences of % Radcliffe Hospital, Oxford OX3 9DU, UK; Dyson many different factors affecting the health and Perrins Laboratory, South Parks Road, Oxford OX1 development of a geographical cohort from pregnancy 3QY, UK onwards. The ALSPAC project is one part of a World Health Organization project taking place in seven Arylamine N-acetyltransferases (NATs) metabolize countries in Europe. The children are examined at arylamines and hydrazines and are usually considered clinics at regular intervals. Additionally, the children as detoxification enzymes. One of two human iso- and their parents are sent questionnaires to gain

further insight into their lives; all this information is transgene is a reflection of its native expression being stored in the ALSPAC database. One aspect of pattern. the project is the ALSPAC DNA bank. DNA samples are being made from blood samples from the children and their mothers. This bank will form a unique Induction of ectopic cartilage by misexpression of Sox9 resource for genetic epidemiology studies and together in chicken embryos with the ALSPAC database will allow many nature versus nurture issues to be examined. About 1000 of CHRIS HEALY, DAFE UWANOGHO and PAUL the children, known as the Children in Focus (CIF), SHARPE are being studied in greater depth than the rest of the Department of Craniofacial Deelopment, UMDS cohort. Some genetic studies on the CIF have been Guy’s Hospital, London Bridge, London SE1 9RT, UK completed. One of these is on mannose-binding lectin (MBL), a protein with a role in innate immunity. The HMG-box DNA binding protein, SOX9, has Mutations in the MBL gene have a semi-dominant been implicated in formation of the embryonic effect and are present in an estimated 40% of the UK skeleton from mutations in the gene in the human population. MBL genotype data, and its relationship disorder campomelic dysplasia, and from its em- to health in childhood in the CIF, will be discussed. bryonic expression pattern. We show that misexpression of Sox9 in io results in ectopic cartilage formation in limbs and in itro is able to change the aggregation properties of limb mesenchymal cells. Fishing upstream of Myf5 Ectopic expression of Sox9 in non-chondrogenic cells also produces ectopic cartilage and results in the OLIVER COUTELLE, DENNIS SUMMERBELL induction of markers of terminal cartilage differen- and PETER W. J. RIGBY tiation. In addition, evidence for a role for Sox9 in Diision of Eukaryotic Molecular Genetics, National ventralization of the somite will be presented. Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

We are seeking to understand how skeletal muscle Patterns of histone H4 acetylation distinguish structural formation is initiated in the mouse by dissecting the and functional domains within the human genome regulatory mechanisms that control the first of the " " myogenic factors, Myf5. In the mouse Myf5 is located COLIN A. JOHNSON , LAURA P. O’NEILL , # 8n5 kb downstream of Mrf4. Previous results have ARTHUR MITCHELL and BRYAN M. " shown that regulatory elements controlling dorsal TURNER " somite expression of Myf5 are contained in the Chromatin and Gene Expression Group, Department intergenic region while ventral expression depends on of Anatomy, The Medical School, Uniersity of # elements in the Myf5 gene itself. Because of the large Birmingham, Birmingham B15 2TT, UK; MRC size of this region we have isolated the Mrf4 and Myf5 Human Genetics Unit, Western General Hospital, genes of the teleost Fugu rubripes, with an 8 times Crewe Road, Edinburgh EH4 2XU, Scotland smaller genome than that of man. Although synteny is conserved in Fugu and the intergenic distance is only The pattern of histone H4 acetylation in different 3 kb, non-coding sequence including the introns was genomic regions has been investigated by immuno- poorly conserved. In contrast, sequence comparison precipitating oligonucleosomes from a human lympho- between the mouse and human Myf5 genes was used blastoid cell line with antibodies to H4 acetylated at successfully to eliminate more than 60% of the intron lysines 5, 8, 12or16. DNA from antibody-bound or sequence and identify conserved regions in the 3h half unbound chromatin was assayed by slot blotting. Pol of each of the Myf5 introns, which together with the I- and Pol II-transcribed genes located in euchromatin 3hUTR can activate reporter gene expression in the were shown to have levels of H4 acetylation at lysines ventral posterior part of the somites. The results 5, 8 and 12 equivalent to those in bulk chromatin, but indicate that multiple elements may be required to to be slightly enriched in H4 acetylated at lysine 16. In control particular anatomical subdomains of Myf5, no case did the acetylation level correlate with actual adding a further level of complexity. Analysis of the or potential transcriptional activity. All acetylated Fugu Myf5 gene in transgenic mice showed remarkable histone H4 isoforms were depleted in non-coding, similarities with the expression pattern of Myf5 in simple-repeat DNA in heterochromatin and the another teleost, the zebrafish Danio rerio. Both are CCCTAA repeat at telomeres, although the extent of expressed in the presomitic mesoderm as well as the depletion varied with the type of heterochromatin and somites, suggesting that the expression of the Fugu with the isoform. Two single-copy genes that map

within or adjacent to blocks of paracentric hetero- in at least four alternatively spliced forms. These are chromatin are depleted in H4 acetylated at lysines 5, deduced to form proteins with two distinct N-terminal 8 and 12, but not 16. Consensus sequences of repetitive regions and the presence or absence of a 50 amino acid elements of the Alu family (SINEs, enriched in R- stretch. These four forms are expressed ubiquitously. bands) were associated with H4 that was more highly The gene (UBE2V) encoding these proteins was acetylated at all four lysines than bulk chromatin, localized to 20q13. There is at least one pseudogene. while H4 associated with KpnI elements (LINEs, Two PACs containing the coding sequence were enriched in G-bands) was significantly under-acetyl- identified. Analysis of these by PCR showed that the ated. sites of possible alternative splicing were coincident with intron–exon boundaries. The exons containing the two first codons and their 5h UTRs were subcloned The T transcription factor functions as a dimer and and initial sequencing results show that they are both exhibits a common polymorphism in the conserved TATA-less. During this time two other proteins DNA binding domain of the human protein similar to E2 enzymes have been described: DDVit1 as a gene whose transcription is induced by vitamin D C. PAPAPETROU, J. C. SOWDEN and Y. H. and Tsg101 as a gene implicated in breast cancer. EDWARDS MRC Human Biochemical Genetics Unit, Uniersity College London, Wolfson House, 4 Stephenson Way, Polymorphism of the lactase gene in different popu- London NW1 2HE, UK lations " " # Using in itro synthesized T protein we have demon- E. J. HOLLOX , M. POULTER , C. B. HARVEY , $ $ " strated that human T binds to its target DNA, 5h- A. KRAUSE , T. JENKINS and D. M. SWALLOW " AGGTGTGAAATT-3h, as a homodimer and that MRC Human Biochemical Genetics Unit, Uniersity truncated protein containing only the N-terminal 233 College London, 4 Stephenson Way, London NW1 # amino acid residues, which comprise the DNA binding 2HE, UK; Unitat de Biologia Cel.lular i Molecular, domain, can form a dimer. We also report a common IMIM, c\o Dr Aiguader, 80, E-08003 Barcelona, $ human polymorphism, Gly177Asp, within the DNA Spain; Department of Human Genetics, South African binding domain at a position which is a conserved Institute of Medical Research and Uniersity of the glycine residue in T homologues from other verte- Witwatersrand, Johannesburg, South Africa brates. This amino acid change appears to decrease the stability of the dimer. It seems possible that T Expression of the small-intestinal enzyme lactase– forms heterodimers with other members of the T-box phlorizin hydrolase (lactase) declines after weaning in transcription factor family. This idea has been most mammals. In humans lactase persists into adult investigated in preliminary studies using Tbx6 protein. life in some people but not others, and this difference We have shown that Tbx6 binds to the same DNA in developmental down-regulation is genetically de- target as T as a dimer, but thus far there is no evidence termined. Allele frequencies vary in different popula- for the formation of heterodimers between T and tions: persistence of lactase into adulthood is common Tbx6. in Northern European populations and pastoral nomadic tribes – both groups that drink milk as part of their diet. It has been shown that the relevant Characterization of a new protein family which re- sequences controlling lactase persistence are cis-acting, sembles E2 enzymes but lacks the catalytic site although the sequence differences have not been identified. However, seven polymorphisms have been CLARE B. HARVEY, ELENA SANCHO and described and in European populations these associate TIMOTHY T. THOMSON to form just four common haplotypes. We will describe Unitat de Biologia Cel.lular i Molecular, IMIM, c\o recent results involving polymorphisms and haplotype Dr Aiguader, 80, E-08003 Barcelona, Spain analysis of various non-European populations, and concentrate on one small highly variable region In a search by RNA differential display for genes upstream from the promoter. This region contains highly expressed in proliferating, as compared with seven variants, four of which have not been published; differentiated, HT29 M6 cells, a cDNA was identified all were characterized by Denaturing Gradient Gel which encodes a protein similar to the E2, ubiquitin Electrophoresis (DGGE) and sequencing. Each conjugating enzymes. However, this protein differs DGGE variant corresponds to a small haplotype from the E2 enzymes in that it does not possess a spanning 84 bases. A model for the evolution of these critical cysteine in the active site. Analysis by Northern haplotypes will be proposed. The region containing blotting and RT-PCRs show that this gene is present variation is otherwise highly conserved in the pig as

well as primates, which suggests a possible functional the identification and characterization of the murine significance. PCM1 gene as well as three additional novel genes (PCMs 2–4) in humans and mice containing MBD- like domains. No further sequence similarity exists The evolution of the murine retroviral restriction gene between any PCM outside of the MBDs with the Fv1 exception of PCMs 2 and 3, which show 78% overall identity at both DNA and amino acid levels. PCMs 2 SCOTT ELLIS, PAUL LE TISSIER and and 3 have been found to bind specifically to JONATHAN STOYE methylated CpGs and are thus candidates for com- MRC National Institute for Medical Research, The ponents of MeCP1. Characterization of this protein Ridgeway, Mill Hill, London NW7 1AA, UK family may provide insights into the mechanism of transcriptional repression by DNA methylation. F1 is a single autosomal gene in mice which determines the resistance or susceptibility to a certain class of mouse retroviruses, the Murine Leukaemia The role of Xist in the regulation of X chromosome Viruses (MLVs). There are two main alleles: the F1n inactivation allele allows infection by N-tropic MLV but restricts B-tropic virus, and the F1b allele allows infection by COLETTE M. JOHNSTON, SARAH M. DUTHIE, B-tropic MLV but restricts N-tropic virus. There are STEVEN A. SHEARDOWN, ALISTAIR E. T. also several other alleles which give rise to slightly NEWALL, EMMA J. FORMSTONE, RUTH M. modified phenotypes. F1 is unique in structure with ARKELL, TATYANA B. NESTEROVA, GIAN- a product which effectively immunizes mice against CARLO ALGHISI, SOHAILA RASTAN and NEIL certain retroviral infections. It is retrovirally derived, BROCKDORFF a remnant of an integration event over 10 million X inactiation Group, MRC Clinical Sciences Centre, years ago which has undergone extreme change. How RPMS, Hammersmith Hospital, London W12 0NN, F1 has changed during the course of evolution has UK been the focus of this work. In order to address this question, F1 genes from many diverse mice, both The onset of X chromosome inactivation in itro and recently- and distantly-diverged members of the genus in io is preceded by a marked increase in the steady- Mus, were cloned and sequenced. The analysis of this state level of Xist RNA. We present data to sequence data has provided considerable information demonstrate that this process is not regulated at the about the events that have led to the evolution of the level of transcription. We show that upregulation can alleles we see today. be accounted for by increased stability of Xist RNA. Using RNA FISH we demonstrate that unstable Xist transcript is produced by alleles both in XX ES cells Identification and characterization of a family of and in XX embryos prior to X inactivation, and that mammalian methyl CpG-binding proteins following differentiation, transcription from the active X chromosome allele continues for a period following BRIAN HENDRICH and ADRIAN BIRD stabilization and accumulation of Xist transcript on Institute of Cell and Molecular Biology, Uniersity of the inactive X allele. Edinburgh, Darwin Building, King’s Buildings, Edin- burgh EH9 3JR, Scotland Imprinted expression of SNRPN in human pre- CpG methylation is usually associated with local implantation embryos transcriptional silencing in eukaryotes. This silencing appears to be mediated through repressors which JOHN HUNTRISS and MARILYN MONK specifically bind to methylated DNA. MeCP1 and Molecular Embryology Unit, Institute of Child Health, MeCP2 are two proteins identified due to their ability 30 Guilford Street, London WC1N 1EH, UK to specifically bind methylated CpGs. Both are capable of repressing transcription in itro. While MeCP2 The SNRPN gene encodes the small nuclear ribo- consists of a single polypeptide, MeCP1 is a large nucleoprotein polypeptide SmN, a 29 kDa spliceo- protein complex. A component of human MeCP1 somal protein expressed predominantly in brain and which binds methylated DNA and is capable of heart. The SNRPN\Snrpn gene is parentally im- repressing transcription has been identified by its printed in human and mouse, being expressed from sequence similarity to the methyl CpG-binding do- the paternal allele only. The human SNRPN gene main (MBD) of MeCP2. This protein was named maps to the smallest deletion region involved in PCM1 for Protein Containing an MBD. We report Prader–Willi syndrome (PWS), a neurogenetic dis-

order, thus implicating the involvement of the gene in some 7 which are imprinted and play a part in the the disease. Imprinting results in a lack of SNRPN aetiology of SRS. We are investigating three growth- expression in PWS cases with paternal deletions or related genes on 7p12–13, a region homologous to a with maternal uniparental disomy (UPD). Further known imprinted region on proximal mouse chromo- cases are caused by disruption of the imprinting centre some 11. The candidates under study are the epidermal (IC), resulting in a failure to establish appropriate growth factor receptor (EGFR) and the insulin-like epigenetic information on the parental SNRPN alleles. growth factor binding proteins 1 and 3 (IGFBP 1 and It is expected that the phenotype induced in disease IGFBP 3). These are all important in growth and are states involving imprinted genes will manifest itself proposed as candidate genes for SRS. We have shown from the time of onset of monoallelic expression, that EGFR is not imprinted in humans using an when no compensatory gene dosage is available from RFLP polymorphism, and are using a sequencing the silent allele. Using single-cell sensitive RT-PCR polymorphism to determine the imprinting status of and a previously reported polymorphism, we have IGFBP 1. We are currently searching for a poly- established that SNRPN expression is monoallelic morphism in IGFBP 3. Further investigation is and expressed from the paternal allele only during planned of genes on chromosome 7 found to be human pre-implantation development. We conclude imprinted. that the epigenetic information necessary for monoallelic expression of SNRPN is already present at this early developmental stage. We will discuss the effect of Developmental effects of genomic imprinting on mouse imprinted SNRPN expression in PWS with respect to chromosome 12 data obtained from murine models, in which a compensatory elevation of the highly homologous PANTELIS GEORGIADES and ANNE C. snRNP protein SmB is observed. There are further FERGUSON-SMITH implications from observations of the distribution of Department of Anatomy, Uniersity of Cambridge, these two proteins within small nuclear ribo- Downing Street, Cambridge CB2 3DY, UK nucleoprotein (snRNP) particles. Thus despite the recent evidence excluding SNRPN as a major factor in The term ‘genomic imprinting’ refers to the fact that PWS, it remains possible that aberrations in SNRPN the two genomes present in an embryo (one inherited expression experienced in PWS are likely to contribute from the mother and the other from the father) are to some of the features of the disease, and that the not functionally equivalent, even though they carry effect will be initiated early in development. the same genetic information. This functional in- equivalence between the two genomes manifests itself as parental-origin-specific differences in expression Search for imprinted genes involved in Silver Russell between the two alleles of certain genes, known as syndrome imprinted genes. Thus, some imprinted genes are expressed from the paternally inherited chromosome " " # S. N. ABU-AMERO , E. L. WAKELING , ,P. and others from the maternally inherited chromosome. " # " STANIER , M. A. PREECE and G. E. MOORE Genetic studies in mice designed to produce offspring " Institute of Obstetrics and Gynaecology, RPMS, in which the parental-origin-specific inheritance of Queen Charlotte’s and Chelsea Hospital, Goldhawk distal chromosome 12 (chr12) was disrupted by having # Road, London W6 0XG, UK; Institute of Child both copies of distal chr12 inherited from the same Health, Uniersity of London, 30 Guilford Street, parent (uniparental disomies or UPDs), strongly London WC1N 1EH, UK suggested that distal chr12 is imprinted and that its correct imprinting is required for normal development. Silver Russell syndrome (SRS) is a dysmorphic However, the phenotype and the cause of death of syndrome associated with intra-uterine growth re- these embryos is not yet known and none of the tardation, short stature, triangular faces and in some published imprinted genes map to chr12. To begin to cases skeletal asymmetry. Maternal uniparental investigate the embryonic role of imprinted genes on disomy for chromosome 7 (mUPD 7) has been chr12, we are carrying out an embryological study to described in approximately 10% of cases of SRS. Five isolate and characterize in detail embryos UPD for cases of mUPD 7 have been found out of 55 SRS chr12 (UPD12). The phenotype of such embryos will patients in our study. All five cases are heterodisomic, be discussed. and using multiple markers across these samples, no areas of common isodisomy have been found. This suggests that the exposure of recessive gene is not the likely cause of the observed phenotype. It is more likely that there are one or more gene(s) on chromo-

Expression of polymorphic murine N-acetyltransferase and recent studies reported at this meeting suggest (NAT2) in CD-1 embryos that this could be due to changes in the placental expression of NAT. In order to determine whether the " # $ $ S. ROLLS , A. J. COPP , J. POPE , E. SIM and embryo itself expresses NAT2, polyclonal anti-NAT2 " L. A. STANLEY antisera [1] were used for immunohistochemical " Department of Pharmaceutical Sciences, De Montfort detection in CD-1 mouse embryos. At 9n5 days post Uniersity, The Gateway, Leicester LE1 9BH, UK; coitum, NAT2 was expressed in the neural tube and # Institute of Child Health, 30 Guilford Street, London yolk sac. By 11n5 days, NAT2 expression was reduced $ WC1N 1EH, UK; Department of Pharmacology, in the neural tube but still strongly evident in the yolk Mansﬁeld Road, Oxford OX1 3QT, UK sac, and by 13n5 days segmental expression of NAT2 was apparent in the developing spinal column. In N-acetyltransferases (NATs) are phase II xenobiotic addition, NAT2 was observed in the brain, eye and metabolizing enzymes. Mice have three Nat genes heart of 13n5 day embryos. These results imply that (Nat1, Nat2 and Nat3), of which Nat2 is known to be both the embryo and its mother must be taken into polymorphic. In addition to its role in xenobiotic account in elucidating the role of NAT in the aetiology metabolism, NAT2 is involved in the clearance of of neural tube defects. folate, a vitamin which protects women at risk of giving birth to oﬀspring with neural tube defects. The 1. Stanley, L. A., Mills, I. G. & Sim, E. (1997). Pharma- rate of folate breakdown increases during pregnancy, cogenetics 7, 121–130.